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Semiquantitative RT-PCR Analysis of Transcripts. To determine the
transcript level in the mutant plants, total RNA was isolated from
the 7-d-old seedlings grown on plates using an RNeasy Mini Kit
(Qiagen). Total RNA (2 μg) was reverse-transcribed using
SuperScript II (Invitrogen) with random primers mixed with oligo
(dT) primers. Equal amount of cDNA was used for PCR with
gene specific primers listed in Table S3. PCR products were analyzed
by ethidium bromide staining.
Anti–γ-Adaptin Antibody Generation. To generate anti–γ-adaptin
antibody, the full-length γ-adaptin was expressed as a fusion
protein with MBP using the pMAL-c2 vector (New England
Biolabs). Recombinant MBP:γ-adaptin was expressed in E. coli
BL21(DE3) strain and purified using amylose resin (New England
Biolabs). MBP:γ-adaptin was used to immunize rabbits and antibody
was affinity-purified using recombinant proteins. To confirm
the specificity of the anti–γ-adaptin antibody, protein extracts
from protoplasts transformed with HA:γ-adaptin were used in
Western blot analysis (1).
Immunohistochemistry. Immunostaining of root tissues was performed
as reported previously (10). In brief, 5-d-old seedlings were
fixed in 4% (wt/vol) paraformaldehyde and labeled with the indicated
antibodies. For immunofluorescence, anti-HA (mouse;
1:1,000; BAbCO), anti-γCOP (rabbit; 1:600; Agrisera), anti-syntaxin
of plants 61 (SYP61) (rabbit; 1:600; a gift from N. Raikhel, University
of Califonia Riverside, Riverside, CA), anti-clathrin heavy
chain (CHC) (rabbit; 1:1,000) (11), anti-KNOLLE (rabbit; 1:4,000)
(10), anti-tubulin (rat; 1:1,000; Abcam), or anti-PIN2 (rabbit, 1:600)
(5) antibodies were used. As the secondary antibody, FITC-labeled
anti-mouse IgG (1:600; Dianova), Cy3-labeled anti-rabbit IgG
(1:600; Dianova), Alexa488-labeled anti-rabbit IgG (1:600; Invitrogen),
or FITC-labeled anti-rat IgG (1:600; Dianova) were used.
Chemical Treatment. Five-day-old seedlings were treated with
50 μM brefeldin A (BFA) (50 mM stock solution in 1:1 DMSO/
EtOH; Invitrogen) or 20 μM wortmannin (20 mM stock solution
in DMSO) for 1 h, followed by fixation or immediate observation.
For FM4-64, 2 μM FM4-64 was added to 5-d-old seedlings
(4 mM stock solution in H 2 O; Invitrogen). For simultaneous
treatment with BFA and FM4-64, BFA was followed by addition
of FM4-64. For BFA washout, BFA-treated seedlings for 1 h
were transferred to new liquid medium and fixed 30 min after the
transfer. For concanamycin A (ConcA) treatment, protoplasts
isolated from WT seedlings were treated at indicated concentrations
(1 mM stock solution in DMSO, Invitrogen). For cycloheximide
(CHX) treatment, 50 μM CHX was added to
seedlings for 1 h (50 mM stock solution in H 2 O; Sigma-Aldrich).
Ultrastructure Analysis. Root tips of 5-d-old seedlings were highpressure-frozen
in 1-hexadecene (Merck Sharp and Dohme)
using a Bal-Tec HPM 010 high-pressure freezer (Balzers). Frozen
samples were freeze-substituted in acetone containing 2.5% (wt/
vol) osmium tetroxide (2 d at −90 °C, 6 h at −60 °C, 6 h at −30 °C,
and 1 h at 0 °C) and finally embedded in epoxy resin (Roth).
Ultrathin sections were stained with 2% (wt/vol) uranyl acetate in
50% ethanol for 10–20 min and lead citrate (3–5 min).
Software. Sequences were analyzed using Vector NTI (Invitrogen)
or CLC DNA Workbench 5. Images were processed using
Adobe Photoshop CS3 and Adobe Illustrator CS3. Quantification
of confocal laser scanning microscopy (CLSM) images was
done using Pearson and Spearman correlation coefficients (PSC)
colocalization plugin of ImageJ [National Institutes of Health
(NIH)] after adjusting the background level to 5 in five independently
taken images according to instructions in the paper
by French et al. (12). Signal intensity in immunoblot analyses
were determined using ImageJ (NIH). Pixel count in live seedlings
was done using Leica LAS in three or four independently
taken images.
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Park et al. www.pnas.org/cgi/content/short/1300460110
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